Protein analysis is the basis of modern biological research. It centers on antigen-antibody interaction to measure levels of antigen of interest under various medical or experimental conditions. An antigen by definition is a foreign molecule that triggers the production of an antibody by the immune system when introduced into the body. The high specificity of the antibody against a specific antigen makes it a powerful tool in clinical, pharmaceutical and biomedical research.
An antigen includes, but not limited to a chemical compound, a peptide, a protein, an RNA, a DNA, a cell (proteins released in situ), or a virus particle (proteins released in situ). The molecule of antigen, as a whole or in part, may be introduced into a host animal, such as a donkey, a goat, or a rabbit to generate a large quantity of antibody against the introduced antigen of interest. Furthermore, the introduced antigen, or part of the antigen, may have more than one epitopes, thus may generate a corresponding number of antibodies against the antigen of interest.
A typical immunodetection process has three major steps. The first step is sample application, in which prepared samples containing an antigen of interest are first bound to a surface, such as nitrocellulose or PVDF membrane or other solid phase like multi-well plate with protein binding capacity. The second step is to form and label the antigen-antibody complex (i.e., immunocomplex) of interest. This step involves the sub-steps of blocking, incubation and washing. In the blocking sub-step, non-specific protein binding sites on the membrane are blocked using a blocking buffer to shield them from non-specific protein. After blocking, the membrane is incubated in the incubation step with antibody against the antigen of interest to form membrane-bound antigen-antibody complex. The unbound antibodies are washed away. The antibody used herein is often commercially available as a pre-labelled antibody. One may also perform the labeling sub-step on site. In either case, the antibody shall be labelled, either directly with a reporter, e.g., a reporter enzyme, or indirectly labelled using a secondary antibody conjugated with a reporter.
The third step is detection. Signals emitted by the reporter enzyme are detected and recorded, which yield information related to the quantity or quality of the immunocomplex bound on the membrane. In both Dot blot analysis and Western blot analysis, the final result of the immunodetection analysis can be further indirectly quantified through densitometric analysis.
There are numerous variations in each individual step of this procedure. For example, the first step—sample application—has many variations, including direct sample application on a piece of membrane in Dot blot analysis, transferring from gel to a piece of membrane in Western blot analysis, or coating of samples to the wells of a microplate in ELISA analysis. Several more modifications have been made to the second step of forming immunocomplexes, including the various procedures and buffer compositions to minimize direct antibody binding while preserving the formed immunocomplex on the membrane. In most cases, the primary antibody is not directly labeled with a reporter enzyme. A secondary antibody against the primary antibody coupled directly or indirectly with a reporter enzyme would be used to label primary antibodies bound to the antigen of interest on the surface of membrane. The secondary antibody may be further labeled with small molecules like Biotin. For example, a Streptavidin coupled HRP may be used to further enhance the signal.
Different methods of labeling the antibody in turn necessitate different corresponding detection methods. For example, the third step of detection may be a color reaction by visual inspection or chemiluminescence signals detectable through scanner, X-ray film or microplate reader, etc. The antibody may also be fluorescence-labeled and detected through scanner
In conventional immunoblot analysis, signals emitted by the labelled immunocomplexes are first acquired as images (e.g., dark bands or dots) through X-ray film or scanner. An image processing equipment (e.g., a densitometer) and/or an image processing software (e.g., ImageJ) is needed to read the image and translates the optical density into a number(s) that indicates the amount of the immunocomplexes.
Among available immunoblotting methods (e.g., dot blot, Elisa, Western blot, reverse phase protein microarray), Western blot probably is the most commonly used in basic lab research. In a Western blot analysis, prepared samples containing the antigen of interest are first separated according to molecular weight through gel electrophoresis, and the separated proteins are transferred through an electroblotting step to either a nitrocellulose membrane or a PVDF membrane.
In an immunodetection process that follows, the levels of the antigen of interest in the prepared samples are detected on the spot in a typical reporter enzyme-based reaction, and quantified indirectly through densitometric analysis. For example, using ImageJ, the bands in the gel image in Western blot analysis are manually selected before calculating numeric values of each band using software. In this process, the specificity of immunodetection is validated by both the antigen-antibody interaction and the expected molecular weight of the antigen of interest, which eliminates false signals commonly observed in Dot blot analysis.
ELISA assay allows direct quantification of signals in a multi-well plate format. However, the protein binding capacity of an Elisa plate is usually of less than 1 μg/cm2. In contrast, a typical membrane for traditional immunoblotting, regardless of nitrocellulose or PVDF membrane, has a protein binding capacity of 100 to 200 μg/cm2. The low binding capacity of ELISA limits its use in the lab.
In short, new and improved methods for analyzing antigen in a complex assay are needed. Suitable methods should be easy to use, inexpensive, fast in assay development, and should yield results comparable to or better than Western blot analysis in measuring the content of antigen in the lysate.